Multiple Sclerosis (MS) is a common disabling disease of young adults. Despite intense immune therapies, relentless progression continues to occur for reasons that are poorly understood. Our overall hypothesis is that Danger/Damage Associated Molecular Patterns (DAMPs) within MS lesions function to inhibit remyelination by preventing the maturation of both oligodendrocyte progenitor cells (OPCs) and premyelinating oligodendrocytes into myelinating oligodendrocytes through activation of the TLR2/MyD88 pathway. We have previously shown that in the presence of DAMPs OPC maturation into myelinating oligodendrocytes is blocked and that in constitutive TLR2 knock-out mice, complete remyelination occurs despite the presence of DAMPs. Our objectives in this application are to provide convincing mechanistic support that DAMPs function in vivo by directly targeting the oligodendrocyte lineage and to identify small molecule antagonists to the TLR2/MyD88 pathway that promote remyelination in MS relevant preclinical models. In the first aim we will compare remyelination in mice harboring cell specific deletions of either TLR2 or MyD88 with strain matched wild-type controls using the chronic cuprizone model. We will assess the impact of TLR2 or MyD88 deletion on the histopathology relevant to chronic demyelination: microglial phenotype, astrogliosis and astrocytic scar, and axonal health. In aim two we will test pharmacologic antagonists to the TLR2/MyD88 pathway for their ability to promote remyelination in MS relevant preclinical models. Using 34 potential TLR2/MyD88 antagonists that we identified in a high throughput screen of pharmacologically active compounds, we will first validate these agents as TLR2/MyD88 antagonists and determine their molecular target in the TLR2/MyD88 pathway. Validated TLR2/MyD88 antagonists will be tested in cerebellar explant organotypic cultures for effectiveness on promoting remyelination. Compounds that induce remyelination in cerebellar explants will then move forward for testing in preclinical animal models. Two models of remyelination will be assessed based on their inherent lack of remyelination under control conditions. The first model is chronic, 12 week, cuprizone, which fails to effectively remyelinate in contrast to the traditional 6-week cuprizone model. The second model is stereotactic focal demyelination with the addition of an MS relevant DAMP, which fails to remyelinate. The value of these models is that they bear clinical relevance to remyelinative failure in MS. Drugs that promote remyelination in these MS relevant preclinical models will then be investigated in greater detail for their pharmacologic targets and their impact on the histopathology of remyelination. We chose to screen a library of pharmacologically active compounds such that agents active in our preclinical models can move forward directly to phase II clinical trail. The unique features of this project are: 1) identification of DAMP signaling and the TLR2/MyD88 pathway as relevant to CNS remyelination; 2) the use of preclinical models that have inherent remyelinative failure; and 3) a clear path forward from preclinical studies to clinical research. !